David N. Fredricks

Breakthrough Fungal Infections in Stem Cell Transplant Recipients Receiving Voriconazole

Infection with voriconazole-resistant fungi may become problematic, because organisms with decreased susceptibility have been noted. Breakthrough fungal infections occurred in 13 of 139 patients who received voriconazole at our center during the period of September 1998 through September 2003. Zygomycetes were found in 6 patients, and Candida glabrata bloodstream infection occurred in 4 patients. Minimal inhibitory concentrations were > or =1 microg/mL for all available isolates. Yeasts and molds with decreased susceptibility to voriconazole may cause invasive infection in patients treated successfully for aspergillosis.

Aspergillus Galactomannan Enzyme Immunoassay and Quantitative PCR for Diagnosis of Invasive Aspergillosis with Bronchoalveolar Lavage Fluid

ABSTRACT Invasive pulmonary aspergillosis (IPA) is frequent and often fatal in hematopoietic stem cell transplant patients. Diagnosis requires microbiological or histopathologic demonstration of the organism in tissues; however, cultivation of Aspergillus species from respiratory secretions has low diagnostic sensitivity. Assays to detect Aspergillus antigen or DNA in bronchoalveolar lavage (BAL) fluid could facilitate earlier diagnosis, thereby guiding optimal therapy and obviating the need for additional costly and potentially morbid diagnostic evaluation. We evaluated the performance of a galactomannan enzyme immunoassay (GM EIA; Bio-Rad) by using a range of index cutoffs to define positivity and a quantitative PCR (qPCR) assay for the detection of Aspergillus species from BAL samples of patients with proven and probable IPA (case patients; n = 49) and without IPA (control patients; n = 50). The sensitivity of the GM EIA was 61% with an index cutoff of 1.0 and 76% with an index cutoff of 0.5; the corresponding specificities were 98 and 94%, respectively. The sensitivity and specificity of qPCR assay were 67 and 100%, respectively. The sensitivity with 22 culture-negative BAL specimens from patients with IPA was 41% for GM EIA with an index cutoff of 1.0, 59% for GM EIA with an index cutoff of 0.5, and 36% for qPCR assay. GM EIA indices and DNA quantities corresponded to BAL fungal burdens, with culture-positive samples having larger amounts of antigen and DNA compared to culture-negative samples. GM EIA and qPCR assay add to the sensitivity of BAL for diagnosing IPA in high-risk patients, with excellent specificity. Adjunctive use of these tests may reduce dependence on invasive diagnostic procedures.

Bacterial Communities in Women with Bacterial Vaginosis: High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria

Background Bacterial vaginosis (BV) is a common condition that is associated with numerous adverse health outcomes and is characterized by poorly understood changes in the vaginal microbiota. We sought to describe the composition and diversity of the vaginal bacterial biota in women with BV using deep sequencing of the 16S rRNA gene coupled with species-level taxonomic identification. We investigated the associations between the presence of individual bacterial species and clinical diagnostic characteristics of BV. Methodology/Principal Findings Broad-range 16S rRNA gene PCR and pyrosequencing were performed on vaginal swabs from 220 women with and without BV. BV was assessed by Amsel’s clinical criteria and confirmed by Gram stain. Taxonomic classification was performed using phylogenetic placement tools that assigned 99% of query sequence reads to the species level. Women with BV had heterogeneous vaginal bacterial communities that were usually not dominated by a single taxon. In the absence of BV, vaginal bacterial communities were dominated by either Lactobacillus crispatus or Lactobacillus iners. Leptotrichia amnionii and Eggerthella sp. were the only two BV-associated bacteria (BVABs) significantly associated with each of the four Amsel’s criteria. Co-occurrence analysis revealed the presence of several sub-groups of BVABs suggesting metabolic co-dependencies. Greater abundance of several BVABs was observed in Black women without BV. Conclusions/Significance The human vaginal bacterial biota is heterogeneous and marked by greater species richness and diversity in women with BV; no species is universally present. Different bacterial species have different associations with the four clinical criteria, which may account for discrepancies often observed between Amsel and Nugent (Gram stain) diagnostic criteria. Several BVABs exhibited race-dependent prevalence when analyzed in separate groups by BV status which may contribute to increased incidence of BV in Black women. Tools developed in this project can be used to study microbial ecology in diverse settings at high resolution.

Targeted PCR for Detection of Vaginal Bacteria Associated with Bacterial Vaginosis

ABSTRACT Several novel bacterial species have been detected in subjects with bacterial vaginosis (BV) by using broad-range PCR assays, but this approach is insensitive for detecting minority species. We developed a series of taxon-directed 16S rRNA gene PCR assays for more sensitive detection of key vaginal bacteria. We sought to determine the prevalence of each species in the vagina, its association with BV, and the utility of PCR for the microbiological diagnosis of BV. Targeted PCR assays were developed for 17 vaginal bacterial species and applied to 264 vaginal-fluid samples from 81 subjects with and 183 subjects without BV. The results were compared to those of two widely accepted methods for diagnosing BV, the use of clinical findings (Amsel criteria) and the interpretation of vaginal-fluid Gram stains (Nugent criteria). Leptotrichia/Sneathia, Atopobium vaginae, an Eggerthella-like bacterium, Megasphaera species, and three novel bacteria in the order Clostridiales are among the bacterial species significantly associated with BV. PCR detection of either a Megasphaera species or one of the Clostridiales bacteria yielded a sensitivity of 99% and a specificity of 89% for diagnosis of BV compared to the Amsel clinical criteria and a sensitivity of 95.9% and a specificity of 93.7% compared to the Nugent criteria (Gram stain). PCR detection of one or more fastidious bacterial species is a more reliable indicator of BV than detection of bacteria, such as Gardnerella vaginalis, previously linked to BV, highlighting the potential of PCR for the diagnosis of BV.

Temporal Variability of Human Vaginal Bacteria and Relationship with Bacterial Vaginosis

Background Little is known about short-term bacterial fluctuations in the human vagina. This study used PCR to assess the variability in concentrations of key vaginal bacteria in healthy women and the immediate response to antibiotic treatment in women with bacterial vaginosis (BV). Methodology/Principal Findings Twenty-two women assessed for BV using Amsels criteria were evaluated daily for 7 or 14 days, then at 2, 3 and 4 weeks, using a panel of 11 bacterium-specific quantitative PCR assays. Participants with BV were treated with 5 days of intravaginal metronidazole. Participants without BV had vaginal biotas dominated by lactobacilli, whose levels fluctuated with menses. With onset of menstruation, quantities of Lactobacillus jensenii and Lactobacillus crispatus decreased and were found to be inversely related to Gardnerella vaginalis concentrations (p<0.001). Women with BV had a variety of fastidious bacteria whose concentrations dropped below detection thresholds 1–5 days after starting metronidazole. Recurrent BV was characterized by initial profound decreases of BV-associated bacteria after treatment followed by subsequent increases at relapse. Conclusions/Significance The microbiota of the human vagina can be highly dynamic. Healthy women are colonized with Lactobacillus species, but levels can change dramatically over a month. Marked increases in G. vaginalis were observed during menses. Participants with BV have diverse communities of fastidious bacteria that are depleted by vaginal metronidazole therapy. Women with recurrent BV initially respond to antibiotic treatment with steep declines in bacterial concentrations, but these bacteria later reemerge, suggesting that antibiotic resistance in these bacteria is not an important factor mediating BV recurrence.

Diagnosis and monitoring of Whipple disease by polymerase chain reaction.

Whipple disease is a systemic bacterial infection with such protean clinical manifestations as diarrhea; abdominal pain; fever; lymphadenopathy; chronic arthralgias; weight loss; and, occasionally, central nervous system involvement that may include dementia, lethargy, and motor and sensory deficits [1-3]. At the time of diagnosis, most patients present with weight loss, diarrhea, and abdominal pain. Recognition of bacillary organisms in a small-bowel biopsy specimen that are positive on periodic acid testing and negative on acid-fast testing leads to the correct diagnosis. However, patients sometimes present with symptoms for which small-bowel biopsy is not usually done. Systemic manifestations, such as pleuritis, lymphadenopathy, cardiac valvular lesions, fever, and wasting may precede gastrointestinal symptoms [1, 2]; symptoms of arthritis may begin years before Whipple disease is diagnosed [2]. Patients with extraintestinal Whipple disease may present with involvement of the central nervous system or the eye [3-6]. Thus, the disease may remain undiagnosed in many patients who never develop gastrointestinal involvement; patients may also receive other diagnoses, such as rheumatoid arthritis or sarcoidosis, in the intervening period. The greatest challenge is to diagnose cases that appear to be clinically consistent with Whipple disease but for which a histologic diagnosis is either negative or equivocal. Because Whipple disease is uncommon, formal prospective evaluations of therapeutic regimens have not been done. Empirical antibiotic therapy with trimethoprim-sulfamethoxazole, cephalosporins, penicillins, or tetracyclines has varied in duration and outcome. In a case series of 29 patients with Whipple disease, the duration of treatment ranged from 5 days to 4 years [2]. Alleviation of symptoms and correction of biochemical and pathologic abnormalities were not related to the duration of antimicrobial therapy. A review of antibiotic therapy in 88 patients with Whipple disease who had long-term follow-up suggested that clinical relapse occurs in as many as 35% of cases [7, 8]; the outcomes of treatment for relapse of disease at the central nervous system were particularly poor [2, 8]. No test for cure is available for Whipple disease, but the recent identification of the 16S ribosomal RNA (rRNA) of the Whipple-associated bacillus, Tropheryma whippelii, has led to the use of 16S rDNA primers for detection of this organism on polymerase chain reaction (PCR) [9, 10]. Polymerase chain reaction assays with high sensitivities have shown T. whippelii in tissues that show no evidence of disease [9]. In this study, a PCR-based test for the detection of T. whippelii was evaluated. Our results suggest that PCR may be useful for the diagnosis of Whipple disease and for monitoring patients who have been treated with antibiotics, thereby allowing the physician to better direct the duration of antibiotic therapy and, perhaps, predict clinical relapse. Methods Patients Thirty patients who received a diagnosis of Whipple disease at the Mayo Clinic between 1952 and 1994 were identified through a computerized search of a diagnosis database. Medical charts of these patients were reviewed. Characteristics of 29 of the patients have been reported elsewhere [2]. Tissue Samples Tissue samples embedded in paraffin blocks were retrieved from the archives of the Mayo Clinic tissue registry; one to seven samples were available from each patient. The anatomic origin of the samples included the small intestine, rectum, pancreas, liver, brain, spleen, and lymph nodes. Post-treatment biopsy specimens were available from 17 of the 30 patients. The medical records of these 17 patients were reviewed by a gastroenterologist who was blinded to the results of PCR and histologic review. All histologic sections were rereviewed in a blinded manner by one pathologist. In small-intestine specimens, the diagnosis of Whipple disease was confirmed on the basis of the following criteria: 1) presence of macrophages in the lamina propria that contained periodic acid-Schiff staining granules, 2) resistance of periodic acid-Schiff-positive granules to diastase, 3) distortion of villous architecture due to the expanded, infiltrated lamina propria, and 4) dilated lymphatic channels (in some instances). Tissue specimens from regions other than the small intestine were also tested for evidence of Whipple disease. Histologic criteria were the presence of foamy histiocytes with bacilli that were positive on periodic acid-Schiff staining and were resistant to diastase and, in tissues from lymph nodes, dilated sinusoids with a Swiss cheese appearance. A histologic section of the small bowel from 1 patient that had been read elsewhere was reexamined in the Mayo Clinics Department of Pathology to confirm the diagnosis; a whole-blood sample (500 micro L) was obtained from this patient after a week of therapy with trimethoprim-sulfamethoxazole. In addition to the 30 patients who had confirmed cases of Whipple disease, 8 patients who had presented with gastrointestinal symptoms and had had biopsy of the small intestine were identified by using a medical records database. Whipple disease had been considered in the differential diagnosis for all 8 patients before biopsy of the small intestine was done. Biopsy specimens were classified as suspicious for or negative for Whipple disease. Medical charts on 7 patients were available for review. Paraffin-embedded biopsy specimens from the small intestine, colon, and brain of 42 patients who had malabsorption of undetermined cause, the irritable bowel syndrome, or viral encephalitis were used as controls. No histologic evidence of Whipple disease was found in these tissues, and all were negative for periodic acid-Schiff-positive macrophages. The control specimens were processed, interspersed with, and tested in a blinded manner in parallel with specimens from the 37 patients described above. Preparation and Analysis of Specimens We extracted two sections of formalin-fixed, paraffin-embedded tissue, each 25 m thick, that had been obtained from each patient. We used a new section of the knife for each cut to avoid cross-contamination during processing. Sections were deparaffinized with xylene and digested overnight at 55 C with 50 L of proteinase K (20 mg/mL) and sodium dodecyl sulfate (10%) in Tris (10 mmol/L) and EDTA (1 mmol/L). The digested tissue was boiled for 15 minutes to inactivate the proteinase K. Deoxyribonucleic acid was extracted by using a chaotropic lysis method with 200 L of the tissue (Isoquick kit, Orca Research, Bothell, Washington). Design of Primers Primers W3FE and W4RB were modified to improve their specificity for T. whippelii [11]. Primers W3AF and W4AR were designed on the basis of 16S rRNA gene sequence alignments of related organisms so that the region of gene that was amplified would be unique to T. whippelii. The expected size of the W3AF and W4AR amplification product was 160 base pairs. Evaluation of Specificity of Primers W3AF and W4AR We determined the specificity of primers W3AF and W4AR by doing PCR on DNA that had been extracted from clinical isolates of many bacteria: Nocardia brasiliensis, N. otitidiscaviarum, N. asteroides, Mycobacterium tuberculosis, M. kansasii, M. chelonae, M. bovis, M. fortuitum, M. avium-intracellulare complex, Actinomyces bovis, A. viscosus, anaerobic Actinomyces species, Corynebacterium species, Propionibacterium species, Rhodococcus equi, unspeciated Rhodococcus, and Streptomyces griseus. Amplification products were found only in R. equi and N. otitidiscaviarum. Sequencing of amplification products of these two organisms showed four variances in nucleotide bases compared with the T. whippelii sequence: C to T at position 1092, G to A at position 1093, C to T at position 1096, and G to A at position 1098. To ensure specific detection of T. whippelii, an 18-mer oligonucleotide probe homologous to T. whippelii sequence was designed to encompass the four-nucleotide variance (see Appendix Table (Table 5)). Table 5. Appendix Table. Sequence of Polymerase Chain Reaction Primers and 16S Ribosomal RNA Positions Relative to Escherichia coli Position 1 Primers W3FE and W2RB, which are also specific for T. whippelii, were used in accordance with the methods described elsewhere [9, 12]. Extraction Control We used the -globin gene, which is found in all nucleated cells, to assess the efficiency of the extraction of DNA from tissue. Tissue samples that were negative for Whipple-associated bacillus 16S rDNA products were amplified for a segment of the human -globin gene. Samples that did not have a positive result when tested for -globin were considered inadequate. Primers GH20 and PCO4 were used for -globin amplification [13]. Polymerase Chain Reaction with Primers W3AF and W4AR An amplification reaction mix, 45 L, was prepared from 10 L of 50% glycerol (10% final concentration), 5 L of 10 x Perkin-Elmer buffer (containing 100 mmol of Tris-HCl per L [pH, 8.3] [Sigma, St. Louis, Missouri], 500 mmol of KCl per L, and 15 mmol of MgCl2 per L) (Applied Biosystems, Perkin-Elmer, Foster City, California), 2 L each of the four deoxyribonucleoside triphosphates, 0.25 units of AmpliTaq polymerase (Applied Biosystems, Perkin-Elmer), 5 pmol of primers W3AF and W4AR, 0.33 L of isopsoralen (25 g/mL), and 19.42 L of sterile water. We added 10% or 5 L of extracted DNA from the specimen to this master mix for a final reaction volume of 50 L. Thermocycling was done as follows. Initial melting was done at 94 C for 2 minutes. This was followed by 50 cycles of denaturing at 94 C for 30 seconds, annealing at 60 C for 45 seconds, and extension at 72 C for 60 seconds. Final extension was done at 72 C for 4 minutes. After each amplification, the microtubes were exposed to ultraviolet light to promote covalent crosslinking of the isopsoralen [14]. This process prevents PCR product contamination, which can

Application of Polymerase Chain Reaction to the Diagnosis of Infectious Diseases

culture reports, whether positive or negative, must be interpreted using an understanding of the test employed and an assessment of the clinical scenario. Similarly, infectious diseases practitioners will need to expand their understanding of PCR-based diagnostics so that these powerful tests are used appropriately. It is our goal to make PCR-based diagnostics understandable to clinicians. We will point out the limitations of conventional diagnostic methods for infectious diseases, discuss the advantages and limitations of PCR-based methods, and mention some current and future applications of this technology. We will not discuss every current or pending application of PCR to diagnostic microbiology; the reader is referred to other publications for additional details [1-3]. We emphasize the principles behind PCR-based diagnosis, and acknowledge a researchoriented bias in our viewpoint. The limitations of existing diagnostic methods and the potential of PCR-based detection and identification methods are demon-

Brief Communication: Fatal Human Metapneumovirus Infection in Stem-Cell Transplant Recipients

Context Human metapneumovirus (hMPV) is a newly discovered respiratory virus. Contribution This study from a cancer referral center retrospectively examined bronchoalveolar lavage samples collected from 1995 to 1999 from hematopoietic cell transplant recipients with new or changing pulmonary infiltrates. The authors cultured hMPV in 5 of 163 symptomatic patients who were thought to have had the idiopathic pneumonia syndrome. Clinical findings included fever, cough, nasal congestion, respiratory failure, pulmonary hemorrhage, and culture-negative sepsis. Four of the patients died. Cautions All patients had moderate to severe symptoms. Implications Human metapneumovirus may cause respiratory failure in immunocompromised adults. The Editors Human metapneumovirus (hMPV) is a newly discovered respiratory virus associated with 10% to 25% of respiratory tract infections in young children (1, 2). This virus has been detected in respiratory disease at all ages (3, 4), although lower respiratory tract disease occurs more frequently in very young and very old persons and in those with underlying conditions. Clinical findings seen with hMPV infection are similar to those of respiratory syncytial virus (RSV) infection (5). As with RSV, serious disease associated with hMPV infection has been reported in immunocompromised patients. Deaths have been reported in a child (5), adults with hematologic malignant conditions (6), and adults after bone marrow or organ transplantation (7, 8). Pneumonia remains one of the most frequent serious complications of hematopoietic stem-cell transplantation (HCT) (9). Despite increased testing for pathogens in the transplantation setting, nearly 10% of myeloablative transplant recipients still develop the idiopathic pneumonia syndrome (10). We retrospectively evaluated archived bronchoalveolar lavage (BAL) fluid from patients undergoing HCT by using a sensitive hMPV-specific, real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay to determine whether hMPV may be a cause of pulmonary morbidity and mortality in this setting. Methods Patients undergoing HCT who underwent BAL between 1995 to 1999 for evaluation of respiratory disease were eligible for inclusion if they had previously consented to diagnostic studies on their tissue and if residual specimens were available and were in good condition. We retrospectively reviewed clinical and laboratory records, biopsy specimens, and autopsy specimens of all patients with detected hMPV. We also evaluated stored BAL samples in 19 HCT recipients who were enrolled in a separate prospective study evaluating BAL after transplantation. Specimens Our institution has used a diagnostic algorithm for defining the cause of pneumonia for patients with new or changing pulmonary infiltrates since 1994 (11). We processed BAL samples in a centralized laboratory for pathogens, as previously described (11). Biopsy and autopsy specimens were submitted for routine bacterial, fungal, and acid-fast bacilli culture tests; underwent cytologic examination; and were tested for mycobacteria, Legionella species, Pneumocystis jiroveci, and fungal species. Additional studies performed in real time included culture and antibody staining of BAL fluid for cytomegalovirus (CMV), RSV, parainfluenza, and influenza viruses. For our study, we assayed BAL from patients with hMPV by using PCR for RSV (12), hMPV (13), parainfluenza types 1 and 3, influenza A and B, adenovirus, human herpesvirus-6 (14), and Aspergillus species (15). In addition, we performed broad-range fungal 18S rDNA PCRs to detect other fungal pathogens (16) and a -globin PCR (17) to determine whether amplifiable DNA was present in BAL fluid. The RT-PCR assay used probes that were designed to amplify 69 and 74 base-pair fragments of the fusion protein genes from the 2 major hMPV groups (13). This assay was sensitive to 10 copies per reaction and was specific for hMPV, as previously described (18). We also examined lung tissue from autopsy specimens of patients with known RSV, CMV, and hMPV infection near the time of death after tissue digestion by using the same RT-PCR methods. Paraffin-embedded tissues were examined for the presence of hMPV antigen by immunohistochemistry in the laboratory of Dr. Thijs Kuiken, Erasmus University, Rotterdam, the Netherlands (19). We included lung sections of an experimentally infected cynomolgus monkey with hMPV as positive controls. Role of the Funding Sources The National Institutes of Health, the Fred Hutchinson Cancer Research Center, and the Adult Leukemia Research Center provided funding for this study. The funding sources had no role in the design, analysis, interpretation, or writing of this manuscript or in the decision to submit the manuscript for publication. Results Patients To determine the presence of hMPV, we evaluated BAL specimens obtained throughout the calendar year from 163 symptomatic and 19 asymptomatic HCT recipients. The mean age of patients undergoing BAL was 42 years; only 10 patients (7%) were younger than 20 years of age. Most patients (99 of 163 patients [60%]) with BAL specimens had received allogenic bone marrow transplants from unrelated donors (57 of 163 patients [35%]), matchedrelated donors (24 of 163 patients [15%]), or mismatchedrelated donors (15 of 163 patients [9%]). Another 64 patients received peripheral blood stem-cell transplants (64 of 163 patients [39%]) from matched allogenic donors (16%), autologous donors (16%), or unrelated donors (4%). One patient received an unrelated cord blood stem-cell transplant. We detected hMPV in the tested BAL specimens of 5 of 163 patients (3.0%) and in 8 of 211 specimens (3.8%). Patients were between 24 and 54 years of age, and they had received different preparative regimens and sources of stem cells (Table). We detected hMPV in samples obtained between January and April. Clinical signs and symptoms around disease onset included a low-grade fever in 4 patients, cough in 3 patients, nasal congestion in 3 patients, and sore throat in 4 patients (although transplantation-related mucositis may have confounded some symptoms). Three patients had wheezing at diagnosis. We did not note sinusitis, otalgia, otitis media, conjunctivitis, or skin rashes. All patients eventually developed radiographic evidence of diffuse pulmonary disease, and we noted rapid progression of pulmonary infiltrates in 4 of 5 patients (Figure, top). Table. Characteristics of Hematopoietic Stem-Cell Transplant Recipients with Human Metapneumovirus Infection Figure. Computed tomography scan of chest in a patient with fatal human metapneumovirus infection after transplantation ( top ) and lung histologic study in a patient with human metapneumovirus pneumonia ( bottom ). Top. Bottom. inset arrow arrow All 5 cases of hMPV lower respiratory tract disease occurred early (<40 days) after transplantation. Lymphopenia was common, although 3 patients had neutrophil engraftment at the time of hMPV detection. Diffuse alveolar hemorrhage was present in 3 patients, and prominent alveolar macrophages were present in another patient. The quantity of hMPV ranged from 2104 RNA copies/mL to 3108 RNA copies/mL of BAL fluid (Table). Detection of Potential Co-Pathogens All BAL sample test results for patients with hMPV were negative for CMV, RSV, influenza viruses, adenovirus, and fungi by real-time PCR. We did not identify any accompanying pathologic or microbiological finding in 3 patients. Herpes simplex virus was isolated at autopsy in case 3, and parainfluenza was detected by culture and PCR in case 4. Case 2 had CMV isolated from lung tissue at autopsy only. We also tested BAL samples from patients with hMPV for human herpesvirus-6. Four of eight sample test results were negative, and the remaining 3 samples had low copies of human herpesvirus-6 DNA (53, 71, and 93 human herpesvirus-6 DNA copies/mL), which are levels compatible with detection of virus in latently infected cells (14). Treatment and Outcome We treated 3 of 5 patients who received a diagnosis of diffuse alveolar hemorrhage (cases 1, 4, and 5) with high-dose corticosteroids without discernable benefit. The hMPV viral load in case 4 decreased despite the use of corticosteroids (Table). Case 2 received aerosolized ribavirin for parainfluenza pneumonia, but additional respiratory samples were unavailable for testing. Progression of respiratory symptoms was rapid, with a median of 4 days (range, 1 day to 32 days) between initiation of oxygen therapy and death. Four patients died of respiratory complications at a median of 16 days (range, 1 day to 35 days) after the BAL was found to be positive for hMPV. Three patients (cases 1, 2, and 3) also had a culture-negative sepsis syndrome that required pressor agents. Clinical diagnoses at the time of death included idiopathic pneumonia in 3 patients; sepsis, shock, or both in 3 patients; and parainfluenza pneumonia in 1 patient. The mortality rate within 3 months of the first BAL in patients with hMPV was significantly higher than that in patients in whom hMPV was not detected (Wilcoxon rank-sum test; P= 0.014). We performed a limited postmortem examination of the lungs for case 2. We noted several bilateral pleural adhesions with diffuse alveolar damage and moderate hemorrhage. The cytologic changes suggested viral infection (Figure, bottom). Postmortem lung culture grew CMV that had not been previously detected. One of 2 archived frozen tissue samples, obtained at autopsy 8 days after bronchoscopy, was found to be positive for hMPV by RT-PCR but was negative by immunohistochemistry. One HCT recipient survived hMPV infection early in her transplantation course. This young patient had neutrophil engraftment when respiratory symptoms were noted. Nonetheless, she experienced a rapidly deteriorating respiratory course and, although she was never intubated, required supplemental oxygen for 2 months. Her hoarseness, dyspnea, and cou

Diversity of Human Vaginal Bacterial Communities and Associations with Clinically Defined Bacterial Vaginosis

ABSTRACT Bacterial vaginosis (BV) is a common syndrome associated with numerous adverse health outcomes in women. Despite its medical importance, the etiology and microbial ecology of BV remain poorly understood. We used broad-range PCR to census the community structure of the healthy and BV-affected vaginal microbial ecosystems and synthesized current publicly available bacterial 16S rRNA gene sequence data from this environment. The community of vaginal bacteria detected in subjects with BV was much more taxon rich and diverse than in subjects without BV. At a 97% sequence similarity cutoff, the number of operational taxonomic units (OTUs) per patient in 28 subjects with BV was nearly three times greater than in 13 subjects without BV: 14.8 ± 0.7 versus 5.2 ± 0.75 (mean ± standard error). OTU-based analyses revealed previously hidden diversity for many vaginal bacteria that are currently poorly represented in GenBank. Our sequencing efforts yielded many novel phylotypes (123 of our sequences represented 38 OTUs not previously found in the vaginal ecosystem), including several novel BV-associated OTUs, such as those belonging to the Prevotella species complex, which remain severely underrepresented in the current NCBI database. Community composition was highly variable among subjects at a fine taxonomic scale, but at the phylum level, Actinobacteria and Bacteroidetes were strongly associated with BV. Our data describe a previously unrecognized extent of bacterial diversity in the vaginal ecosystem. The human vagina hosts many bacteria that are only distantly related to known species, and subjects with BV harbor particularly taxon-rich and diverse bacterial communities.

Randomized, Placebo-Controlled Phase 2 Trial of a Lactobacillus crispatus Probiotic Given Intravaginally for Prevention of Recurrent Urinary Tract Infection

BACKGROUND Urinary tract infections (UTIs) are common among women and frequently recur. Depletion of vaginal lactobacilli is associated with UTI risk, which suggests that repletion may be beneficial. We conducted a double-blind placebo-controlled trial of a Lactobacillus crispatus intravaginal suppository probiotic (Lactin-V; Osel) for prevention of recurrent UTI in premenopausal women. METHODS One hundred young women with a history of recurrent UTI received antimicrobials for acute UTI and then were randomized to receive either Lactin-V or placebo daily for 5 d, then once weekly for 10 weeks. Participants were followed up at 1 week and 10 weeks after intervention and for UTIs; urine samples for culture and vaginal swabs for real-time quantitative 16S ribosomal RNA gene polymerase chain reaction for L. crispatus were collected. RESULTS Recurrent UTI occurred in 7/48 15% of women receiving Lactin-V compared with 13/48 27% of women receiving placebo (relative risk [RR], .5; 95% confidence interval, .2-1.2). High-level vaginal colonization with L. crispatus (≥10(6) 16S RNA gene copies per swab) throughout follow-up was associated with a significant reduction in recurrent UTI only for Lactin-V (RR for Lactin-V, .07; RR for placebo, 1.1; P < .01). CONCLUSIONS Lactin-V after treatment for cystitis is associated with a reduction in recurrent UTI. Larger efficacy trials of this novel preventive method for recurrent UTI are warranted. CLINICAL TRIALS REGISTRATION. NCT00305227.